Interpretive Summary: A severe outbreak of a foliar decline disease was observed in the mulberry orchards at Hangzhou city, Zhejiang province, China in August, 2006. Typical symptoms of the disease are dark brown discolorations in vascular tissues, leaf wilt, defoliation, and tree decline. Unlike the bacterial wilt disease caused by Ralstonia solanacearum, the leaf wilt symptoms generally started from the bottom of the plants and moved upward. Based on Biolog metabolic profiles, fatty acid methyl ester analysis (FAME) and sequence analysis of the partial 16S rDNA and rpoB gene of the bacteria, the causal agents of the disease were identified as a complex of Enterobacter spp. Characterization of four selected isolates revealed that two of them, isolates R2-2 and R6-2, belonged to E. asburiae, and the other two isolates, R11-2 and R18-2, should be classified as a new species of the genus Enterobacter. The identification and characterization of the new disease revealed the complexity of the casual agents, and provided a useful tool anf information for diagnostics and disease management.

Technical Abstract:
A new mulberry wilt disease (MWD) was recently identified in Hangzhou, Zhejiang province, China. Typical symptoms of the disease are dark brown discolorations in vascular tissues, leaf wilt, defoliation, and tree decline. Unlike the bacterial wilt disease caused by Ralstonia solanacearum, the leaf wilt symptoms generally started from the bottom of the plants and moved upward. Based on Biolog metabolic profiles, fatty acid methyl ester analysis (FAME) and sequence analysis of the partial 16S rDNA and rpoB gene of the bacteria, the causal agents of the disease were identified as a complex of Enterobacter spp. Four MWD isolates were purified and their Biolog profiles identified them as belonging to the genus Enterobacter. Three of the isolates were identified as E. cloacae with similarity indices ranging from 0.56 to 0.65. The fourth isolate, R11-2, had a high index level (0.73) to E. aerogenes. Comparisons of the four isolates’ fatty acid profiles with those in the MIDI database revealed the similarity indices of the “closest” matches were in the species of E. cancerogenus and the mean closest match was 0.70. The 16S rDNA sequences of isolates R2-2 and R6-2 shared a 98.1% and 97.7% identity to type strain E. cancerogenus LMG2693, respectively, and a 98.3% and 97.2% to type strain E. asburiae JCM6051, respectively. The relationship between isolates R2-2 and R6-2 and the type strain E. asburiae JCM6051 was confirmed by the rpoB gene sequence. The isolates showed a similarity of 98.4% and 99.0%, respectively, to E. asburiae JCM6051. No 16S rDNA sequence similarity above 97% was found between either of the isolates, R11-2 or R18-2, and any recognized species, suggesting that the two isolates might represent one or two novel Enterobacter species. Their rpoB gene similarity values, when compared to their nearest type strains, were lower than 98%, providing further evidence that the two isolates represent novel species within the genus Enterobacter.